The long term objective of this proposal is to better understand the molecular structure and mechanism of catalysis by two multinuclear manganese enzymes which share structural and functional homologies. Manganese catalases are novel (non-heme type) catalases that serve to protect against oxidative damage caused by hydrogen peroxide, a potent biological toxin, by conversion to water and oxygen. The photosynthetic water oxidizing enzyme is a complex multisubunit protein associated with a photochemical reaction center protein. It contains a tetramanganese cluster along with Ca2 plus and Cl ions needed to catalyze the oxidation of water to oxygen. Catalysis of this chemistry is so complex that only a single type of enzymatic site appears to have evolved0 in Nature. The health of all aerobic organisms, including man, depends upon this process, since it is the dominant renewable source of atmospheric oxygen. Manganese catalase from the extreme thermophile, Thermus thermophilus, will be compared to the water oxidase from spinach and a cyanobacterium. The proposed methods and specific goals are: 1) two new intermediates formed during assembly of the water oxidase from its apo- protein and inorganic reagents (Mn2 plus, Ca2 plus, Cl) have been discovered. These will be kinetically characterized using methods that are capable of revealing the number of inorganic ions bound, their oxidation states, thermodynamic binding constants and 1H/2H isotopic rate dependences. This will be accomplished using two newly developed instruments capable of ultrasensitive detection of O2 and protons, and by spectrophotometry; 2) the sites of binding of these cofactors within the protein and the intercofactor interactions (electrical field gradient at Mn and magnetic dipolar and scalar couplings) will be determined using magnetic resonance spectroscopies (EPR, ESE-ENDOR and ESEEM) of isotopically labelled protein samples and cofactors; 3) assembly of functional "inorganic mutants", for example, by replacement of Ca2 plus with Sr2 plus and Mn2 plus by Ru2 plus, among others, will be examined in an effort to catalyze new reactions or to accumulate partially assembled intermediates; 4) the mechanism of inhibition and catalysis by Mn catalase will be examined at the atomic level by detection of the reactive intermediates formed during peroxide dismutation using rapid mixing spectrophotometry, transient FT-EPR, resonance Raman and NMR spectroscopies in Princeton and using X-ray crystallography by our collaborator Dr. V. Barynin; 5) substrate 2H/1H and 18O/16O isotope effects will be measured to characterize the degree of bond making and breaking in the transition state; 6) characterization of site-directed mutants of Mn catalase will begin with a focus on creation of monomeric protein subunits for structural analysis by NMR.